Culture based screening assay and methods of use thereof to identify agents which modulate tumor development, invasion and differentiation

Abstract

A 3D organotypic culture which phenocopies aggressive, invasive cancer and methods of use thereof are provided.

What is claimed is: 
     
       1. A method for in vitro screening of a compound comprising:
 a) providing an organotypic culture comprising an artificial stroma of collagen and human fetal esophageal fibroblasts, said fibroblasts comprising an inducible AKT gene, and being overlayed with
 i) esophageal keratinocytes transduced with at least one activated oncogene and a transduced nucleic acid which inhibits p120ctn action selected from the group consisting of small hairpin RNA, siRNA and microRNA, or 
 ii) keratinocytes present in an esophageal tumor isolated from a patient; 
 
 b) contacting said cultures with said compound and an AKT inducing agent, AKT induction producing a more invasive, less differentiated cancer phenotype; 
 c) observing the effect of said compound upon migration and invasive characteristics of said transduced esophageal keratinocytes or esophageal tumor keratinocytes; and 
 d) determining whether said compound modulates expression levels of periostin. 
 
     
     
       2. The method of  claim 1 , wherein said at least one activated oncogene of step (a)(i) is cyclin D1. 
     
     
       3. The method of  claim 1 , further comprising assessing the effect of said compound on cultures which do not contain said nucleic acids. 
     
     
       4. The method of  claim 2 , wherein said compound is an MMP-9 inhibitor and inhibits tumor cell migration and invasion. 
     
     
       5. The method of  claim 2 , wherein said compound is an AKT inhibitor. 
     
     
       6. The method according to  claim 1 , wherein said compound is screened for toxicity to human tissue, and the effects of said compound on the morphology and life span of said keratinocytes is determined, thereby identifying a compound which reduces the life span of the cells or has a negative impact on the morphology of said cells. 
     
     
       7. The method of  claim 1 , wherein expression of hepatocyte growth factor (HGF) is determined. 
     
     
       8. The method of  claim 1 , wherein said culture further comprises human smooth muscle cells embedded in said collagen. 
     
     
       9. The method of  claim 1 , wherein said culture further comprises a layer of endothelial cells underlying said collagen and fibroblast mixture, where said epithelial cells form a capillary network induced by fibroblasts in said mixture.